Method of purifying polypeptides by simulated moving bed chromatography

ABSTRACT

Provided are methods of separating an immunoreactive compound from at least one immaterial component, using a simulated moving bed (“SMB”) system and a SMB apparatus for use in these methods. Also provided are purified immunoreactive compounds prepared using the SMB methods and apparatus and methods of treatment with the purified immunoreactive compounds.

FIELD OF THE INVENTION

[0001] The present invention is directed to methods of purifyingpolypeptides using simulated moving bed chromatography and to simulatedmoving bed chromatography systems and apparatus suitable for purifyingpolypeptides.

BACKGROUND AND INTRODUCTION TO THE INVENTION

[0002] Chromatographic separations used for protein purification aretraditionally performed in batch mode, i.e. a single packed column isused and equilibration, load, wash, elution and regeneration/cleaningare performed sequentially. This mode leads to a comparatively longprocess with fairly low overall throughput. In addition, due to kineticlimitations of protein adsorption, in a batch mode, chromatographycolumns are loaded only to their so-called dynamic capacity whichusually is 30 to 50% of their equilibrium capacity. This in turnrequires using columns of two to three times the volume than would beneeded if the columns were operated in equilibrium. Since proteinchromatography resins are very expensive, this has major economicconsequences adding to the cost of purification of product.Additionally, wash and elution processes in batch column chromatographyrequire substantial fluid volumes, which is also due to the batch modeof operation which has economic consequences since the purified waterused must be specially treated.

[0003] Simulated Moving Bed (SMB) chromatography has been used in thepetrochemical and mineral industries. SMB chromatography has also foundapplication in the pharmaceutical industry for the separation ofenantiomers.

[0004] Other applications of SMB chromatography include, for example,the separation of fructose from fructose-glucose solutions and theseparation of sucrose from sugar beet or sugar cane syrups. Solutioncomponents are differentially absorbed by the ion exchange resin so thata separation waveform develops within the simulated moving bed.

SUMMARY OF THE INVENTION

[0005] The present invention is directed to methods of purifyingimmunoreactive proteins by separation from at least one immaterialcomponent using simulated moving bed (“SMB”) chromatography, to SMBsystems and to apparatus useful in these methods, to immunoreactiveproteins separated by SMB chromatography and to methods of treatingpatients with the separated immunoreactive proteins.

[0006] In one aspect, the present invention is directed to a method ofseparating an immunoreactive compound from at least one immaterialcomponent in a fluid mixture using simulated moving bed chromatography.According to this aspect, the method comprises the steps of: (a)providing a simulated moving bed apparatus that comprises a plurality ofmodules in fluid conducting communication, said modules comprising atleast one solid phase; (b) continuously introducing the fluid mixtureinto said simulated moving bed apparatus wherein the fluid mixturecontacts the solid phase in a countercurrent mode; (c) effectingseparation of the immunoreactive compound from at least one immaterialcomponent; and (d) collecting the immunoreactive compound to provide apurified composition thereof. According to one embodiment of thisaspect, the immunoreactive compound associates with the solid phase to agreater or lesser degree than at least one immaterial component.Preferably, the immunoreactive compound associates with the solid phaseto a greater degree than at least one immaterial component. According toan alternate embodiment, this method further comprises the step ofeffecting said separation by contacting the solid phase with an eluentthat promotes disassociation of the immunoreactive compound. Suitableeluents include an acidic buffer. Suitable solid phases include asupport material associated with Protein A or Protein G. The separationmay be effected using chromatographic methods such as adsorptionchromatography, partition chromatography, ion exchange chromatography,size exclusion chromatography or affinity chromatography. Preferably,separation is effected using affinity chromatography. The presentinvention is also directed to an immunoreactive compound preparedaccording to these methods.

[0007] In another aspect, the present invention is directed to asimulated moving bed (“SMB”) system for separating an immunoreactivecompound from at least one immaterial component when both are present ina fluid mixture. According to this aspect the SMB system incorporates aplurality of zones comprising a solid phase which is contactedcountercurrently by said fluid mixture and wherein said zones comprise(i) an association zone wherein the immunoreactive compound and at leastone immaterial component differentially associate with the solid phase;(ii) a wash zone wherein at least one immaterial component ispreferentially disassociated from the solid phase; and (iii) an elutionzone wherein the immunoreactive compound is preferentially disassociatedfrom the solid phase. According to one embodiment, the wash zone isintermediate between the association zone and the elution zone. Thesolid phase may preferentially associate with the immunoreactivecompound. According to one aspect of this embodiment, the solid phasecomprises a ligand for affinity chromatography. Suitable solid phasesmay comprise Protein A or Protein G. An alternate suitable solid phasecomprises a cation exchange resin. According to one aspect of thisembodiment, the simulated moving bed system further comprises an elutionwash zone intermediate between the elution zone and the associationzone. Also, this embodiment may further comprise a regeneration zone andintermediate between the elution wash zone and the association zone andalso an equilibration zone intermediate between the regeneration zoneand the association zone. The present invention is also directed to amethod of separating an immunoreactive compound from at least oneimmaterial component which comprises using simulated moving bed systemsand to a purified immunoreactive compound prepared using such simulatedmoving bed systems.

[0008] According to a further aspect, the present invention is directedto a method of separating an antibody from at least one immaterialcomponent in a fluid mixture where both are present using simulatedmoving bed chromatography which comprises the steps of (a) providing asimulated moving bed apparatus that comprises a plurality of modules influid conducting communication, said modules comprising at least onesolid phase which comprises an affinity chromatography ligand whichpreferentially associates with the antibody; (b) continuouslyintroducing the fluid mixture into the simulated moving bed apparatuswherein the fluid mixture contacts the solid phase in a countercurrentmode; (c) effecting separation of the antibody from at least oneimmaterial component; and (d) collecting the antibody to provide apurified composition thereof.

[0009] In an additional aspect, the present invention is directed to asimulated moving bed (“SMB”) system for separating an antibody from atleast one immaterial component where both are present in a fluidmixture. According to this aspect, the SMB system incorporates aplurality of zones comprising a solid phase comprising an affinity resinwhich is contacted countercurrently by the fluid mixture and whereinsaid zones comprise: (i) an association zone wherein the antibody and atleast one immaterial component differentially associate with the solidphase; (ii) a first wash zone wherein at least one immaterial componentis preferentially dissociated from the solid phase; and (iii) an elutionzone wherein the antibody is preferentially disassociated from the solidphase.

[0010] In another aspect, the present invention is directed to a methodof separating an immunoreactive compound from at least one immaterialcomponent in a fluid mixture using simulated moving bed chromatographycomprising the steps of: (a) providing a simulated moving bed apparatuswhich comprises at least one module in fluid conducting communicationwith said apparatus, said module comprising at least one solid phase andwherein said apparatus comprises a plurality of zones through which themodules pass; (b) continuously introducing the fluid mixture into themodule in an association zone wherein the fluid mixture contacts thesolid phase in a countercurrent mode and wherein the immunoreactivecompound associates with the solid phase; (c) continuously introducing awash buffer into the module comprising the associated immunoreactivecompound in a wash zone wherein the wash buffer contacts the solid phasein a countercurrent mode and substantially removes at least oneimmaterial component from said module; (d) continuously introducing anelution buffer into the module comprising the associated immunoreactivecompound in an elution zone wherein the elution buffer contacts thesolid phase in a countercurrent mode and whereby the immunoreactivecompound is substantially disassociated from the solid phase; and (e)continuously removing a product stream comprising the immunoreactivecompound from the module. According to one embodiment, the simulatedmoving bed apparatus comprises a plurality of modules. Preferably, solidphase comprises an affinity ligand. Suitable affinity ligands includeProtein A or Protein G. According to one aspect, the immunoreactivecompound comprises an antibody or antibody fragment. The presentinvention is also directed to a purified antibody or antibody fragmentprepared substantially by these methods.

[0011] According to another aspect, the present invention provides animproved method of purifying an immunoreactive compound from at leastone immaterial component, the improvement which comprises usingsimulated moving bed affinity chromatography with a solid phasecomprising Protein A or Protein G.

[0012] Suitable immunoreactive compounds to be separated using the SMBmethods of the present invention include antibodies and antibodyfragments. Such immunoreactive compounds include those which bind to anantigen selected from the group consisting of CD20, CD40, CD40L, CD23,CD4, CD80 and CD86.

[0013] Definitions

[0014] In accordance with the present invention and as used herein, thefollowing terms are defined to have the following meanings, unlessexplicitly stated otherwise:

[0015] The term “immunoreactive compound” refers to a compound whichcomprises an antigen binding region and/or a constant region from animmunoglobulin. In one aspect, included is a compound which binds to anantigen. An immunoreactive compound may additionally comprise peptidesequences from non-antibody compounds, such as cytokines, cytokineligands and other antigens. Such immunoreactive compounds includeantibodies, antibody fragments, domain-deleted antibodies, or anantibody linked to another specific binding member or mixtures thereof.Domain-deleted antibodies include compounds such as those described inWO 02/060995, the disclosure of which is incorporated herein byreference. Other immunoreactive compounds include fusion proteins whichcomprise a region of an immunoglobulin chain fused to an amino acidsequence such as an amino acid sequence of a ligand binding partner oran amino acid sequence variant of an adhesion. Such fusion proteinsinclude those described in U.S. Pat. Nos. 5,428,130 and 5,565,335, thedisclosures of which are incorporated by reference herein.

[0016] The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, humanized antibodies, primatizedantibodies, domain-deleted antibodies, and antibody fragments so long asthey exhibit the desired biological activity.

[0017] “Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)Z, and Fvfragments; diabodies; linear antibodies; single chain antibodymolecules; and multispecific antibodies formed from antibody fragments.Domain deleted antibodies comprising immunoglobulins in which at leastpart of one or more constant regions have been altered or deleted toprovide modified physiological properties (e.g. reduced serum half-life)may also be considered antibody fragments for the purposes of theinstant disclosure. In preferred embodiments the domain deletedantibodies will comprise constant regions that lack the C_(H)2 domain.

[0018] “Chromatography” refers to any analytical technique used for thechemical separation of mixtures and components, that relies uponselective attraction among the components of a mixture for a solidphase. Examples include adsorption chromatography, partitionchromatography, ion exchange chromatography, size exclusionchromatography, and affinity chromatography.

[0019] “Adsorbent” is used herein generically to refer to the solidphase used in chromatography for which the mobile phase componentsexhibit a selective affinity. Because such affinity can take a varietyof forms other than adsorption (including size exclusion orcomplexation), the term refers to solid phases that adsorb thecomponents of a mixture and to solid phases that do not technicallyadsorb components from the mobile phase, but which nevertheless behaveas an adsorbent by slowing the migration velocity of one componentrelative to another in a chromatographic system.

[0020] “Purified” when referring to a component or fraction indicatesthat its relative concentration (weight of component or fraction dividedby the weight of all components or fractions in the mixture) isincreased by at least 20%. In one series of embodiments, the relativeconcentration is increased by at least 40%, 50%, 60%, 75%, 100%, 150%,or 200%. A component or fraction can also be said to be purified whenthe relative concentration of components from which it is purified(weight of component or fraction from which it is purified divided bythe weight of all components or fractions in the mixture) is decreasedby at least 20%, 40%, 50%, 60%, 75%, 85%, 95%, 98% or 100%. In stillanother series of embodiments, the component or fraction is purified toa relative concentration of at least 50%, 65%, 75%, 85%, 90%, 97%, 98%,or 99%. When a component or fraction in one embodiment is “separated”from other components or fractions, it will be understood that in otherembodiments the component or fraction is “purified” at levels providedherein.

[0021] “Module” refers to a portion of a simulated moving bed apparatus.A module may comprise one or a plurality of columns or vessels.

[0022] “Ring” is used to describe how the modules of a SMB system areconfigured in relation to one another in the SMB system because theoutput of each module comprises the input for the successive module, ina circular fashion. The term “ring” should thus not be understood to belimited to a circular configuration of the modules and columns withinthe modules.

[0023] “Multicomponent mixture” refers to a fluid mixture that comprisesthree or more components or fractions which can be separated using aprescribed chromatographic process, because each component or fractiondisplays a different affinity for the adsorbent employed.

[0024] “Immaterial component” refers to a component present in the fluidmixture containing the immunoreactive compound which is not animmunoreactive compound and which is separated from immunoreactivecomponent. Immaterial components may include host cell protein (HCP),antibiotics and other components present in the fluid mixture.

[0025] “Mass transfer effects” refer generally to those physicalphenomena which cause components of a mixture to display distinctdispersion behavior from the mixture in a given system, and to departfrom the ideal system. Mass transfer effects thus include those effectsmodeled using axial dispersion coefficients, intraparticle diffusioncoefficients, and film mass transfer coefficients. Mass transfer effectsthus also include fronting and dispersion due to extra-column deadvolume. A separation is hindered by non-negligible mass transfer effectsif the mass transfer correction term, discussed in more detail below, ismore than 2% of the mobile phase velocity in any of the zones asprescribed by equations 3-6 (the mobility phase velocities for an idealsystem), to achieve a prescribed purity and yield. The designs of thepresent invention can also extend to systems in which the mass transfercorrection velocity increases or decreases the mobile phase velocity forthe ideal system by more than 1, 3, 5, 7.5, 10, 15, 20, 30, 50, 75,100%, 200%, 400%, 600%, 1000%, or more.

[0026] As used herein, “substantially separated from other components”means that the separated component contains no more than about 20% byweight of each other component, preferably no more than about 5% byweight of each other component, and more preferably no more than about1% by weight of each other component.

[0027] As used herein, “a stream that does not contain substantialamounts of a component” means that the stream contains at most about 20%by weight of the component, preferably at most about 5% by weight of thecomponent, and more preferably at most about 1% by weight of thecomponent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 depicts a flow chart of a general procedure for isolationand purification of an immunoreactive compound, such as an antibodyproduced in cell culture.

[0029]FIG. 2 depicts a flow diagram for an alternate general procedurefor isolation and purification of an immunoreactive compound such as anantibody from cell culture.

[0030]FIG. 3 depicts a diagram of a four (4) zone simulated moving bedchromatography (SMB) system of the present invention.

[0031]FIG. 4 depicts a diagram of an embodiment of a SMB systemaccording to FIG. 3.

[0032]FIG. 5 depicts a diagram of an alternate four (4) zone SMB systemof the present invention.

[0033]FIG. 6 depicts a diagram of an embodiment of a SMB systemaccording to FIG. 5.

[0034]FIG. 7 depicts a diagram of an eight (8) zone SMB system of thepresent invention.

[0035]FIG. 8 depicts a diagram for an eight (8) zone simulated movingbed system of the present invention which has twenty (20) columns. FIG.8 represents a two dimensional depiction of a carousel apparatus whereinthe columns are arranged in a circle.

[0036]FIG. 9 depicts a diagram for an alternate embodiment of an eight(8) phase simulated moving bed system of the present invention which hastwenty (20) columns.

[0037]FIG. 10 depicts a diagram for an alternate embodiment of an eight(8) phase simulated invention which has twenty (20) columns.

[0038]FIG. 11 depicts a UV trace from Wash 1 for a series of twenty (20)column switches within one rotation of the SMB apparatus depicted inFIG. 9 as described in Example 3.

[0039]FIG. 12 depicts a plot of antibody concentration (IgG titer), IgGflux and concentration of host cell proteins (HCP) for nine (9) fullrotations of the SMB system depicted in FIG. 9. In this figure the topplot of diamonds (♦) depicts IgG flux; the lower plot of diamonds (♦)depicts IgG titer; and the plot of the squares (▪) depict HCP

DETAILED DESCRIPTION OF THE INVENTION

[0040] According to one aspect, the present invention is directed tosimulated moving bed (“SMB”) systems and to simulated moving bedchromatography methods suitable for use in a process of isolating andseparating immunoreactive compounds, such as antibodies made in cellculture. The SMB systems and methods of the present invention may beused in a process such as that depicted in FIG. 1 for either Step D orStep F or FIG. 2 for either Step B or D.

[0041] These processes typically incorporate one or more chromatographysteps. Since protein chromatography resins are very expensive, the useof chromatography columns in a batch mode loaded only to their so-calleddynamic capacity (which usually is 30 to 50% of their equilibriumcapacity), requires using columns of two to three times the volume thanwould be needed if the columns were operated in equilibrium (i.e.,loaded to their equilibrium capacity). Thus, use of a batch mode for thechromatography steps has major economic consequences. Additionally, washand elution processes in batch column chromatography require substantialfluid volumes, which is also due to the batch mode of operation. Use ofcontinuous chromatography, in a continuous, countercurrent mode wherethe columns are loaded to equilibrium capacity would require smallercolumn volumes. Additionally more efficient wash and elution processeswould lead to substantially reduced consumption of buffers. One way ofputting this into practice is use simulated moving bed chromatography.

[0042] A typical simulated moving bed system has at least one module ora plurality of modules filed with solid phase. A module may include oneor a plurality of columns or vessels. Fluid conduits interconnect theupstream and downstream ends of the system to form a loop through whicha fluid mixture is continuously circulated. At certain points liquidstreams may be introduced and at other points effluent streams may bewithdrawn. The constant flow of fluid through the loop is called“internal recirculation flow.” A manifold system of pipes and valves isprovided selectively to position an inlet for feed material, an inletfor elution buffer (to disassociate a component from the solid phase),an outlet for a disassociated component and an outlet for anunassociated (or less associated) component. Each inlet and outletcommunicates with a separate module (or vessel or column). Feed materialenters the system at a designated point and is moved through the solidphase by the continuous internal recirculation flow. This moving contactresults in a chromatographic separation of components. Unassociatedcomponent(s) which flow(s) at a relatively fast rate are removed from anunassociated component outlet, such as by removal of a first washeffluent stream. A buffer which disassociates an associated compoundfrom the solid phase (elution buffer) is added at its inlet valuebetween the respective outlet valve positions of the associated andunassociated components.

[0043] At predetermined time intervals (switch time) the designatedinlet and outlet valve positions are displaced downstream one positionon the manifold to the next solid phase bed module, which may be adiscrete section of a vessel, (such as a column), or an individualvessel, e.g., column. The step time is chosen such that the designationof valves is properly synchronized with the internal recirculation flow.Under these conditions the system eventually reaches a steady state withspecific product characteristics appearing at predetermined intervals insequence at each valve position. This type of system simulates valvesheld in a single position while the solid phase moves at a constant andcontinuous rate around the recirculation loop producing constant qualityproduct at each valve. An alternative apparatus actually intermittentlymoves the columns—often mounted on a carousel—while the valve locationsare fixed.

[0044] The simulated version more closely approaches the character of anactual moving bed system as the number of modules (or vessels orcolumns) and valve positions increase. An important distinction betweenbatch and simulated moving bed system is that the internal recirculationflow is continuous in the simulated moving bed process. Except for verysmall adjustments to control internal pressure, the entering and exitingflow rates are continuous and constant, thereby approximating an actualmoving bed system as closely as possible.

[0045] An equilibrated SMB system exhibits a steady state componentseparation waveform along the path of the recirculation loop. Thiswaveform moves along the path of the recirculation loop with valveswitching synchronized to maintain the desired steady state. In such aSMB system the zones may be viewed as stationary with the modules movingthrough the zones.

[0046] Simulated moving bed processes realize the countercurrentmovement of solid and liquid phases and the concomitant advantages ofcontinuous moving beds over batch chromatography without the physicalmovement of solids. SMB processes utilize a series of modules comprisingat least one solid phase connected to form a circuit. In some systems,each module contains at least one or two or more evenly sized columns,and these columns are connected to form a continuous circuit. The solidphase movement is simulated by periodically moving the inlet and outletports one module (of if the module comprises a plurality of columns, onecolumn) forward in the direction of flow of the mobile (fluid) phase, sothat the product ports are always near the partially separatedconcentration waves of products in the system. Similar to the continuousmoving bed system, the port switching time, zone length, and zone flowrates are all balanced to attain a desired level of purity of theproduct stream. Thus, the zones may be viewed as stationary with themodules moving through one zone to another.

[0047] 1. General Immunoreactive Protein Separation Processes

[0048] The SMB methods of the present invention are useful as part of amethod for the separation of immunoreactive compounds, such asantibodies made by cell cultures, from immaterial component(s).

[0049]FIG. 1 depicts a schematic for a general process for theseparation and isolation of immunoreactive compounds such as antibodiesproduced by cell culture from at least one immaterial component. Themethods of the present invention may be used as a separation step inthis general method or as a separation step in other general processesused in the art to isolate and purify immunoreactive compounds such asantibodies produced by recombinant and cell culture methods.

[0050] According to the process depicted in FIG. 1, the cell culturesuspension in Step A is treated to harvest cells. In Step B, the cellculture suspension is clarified (cells, cell debris and precipitates areremoved). One suitable method for step B is depth filtration; othersuitable methods include centrifugation and tangential flow filtration.In Step C the filtrate is incubated with Triton X-100, suitably in avessel such as in a holding tank. Treatment with Triton X-100 isbelieved to inactivate viruses which may be present. In Step D, thefiltrate containing at least one immaterial component, such as host cellprotein (“HCP”), and immunoreactive compound undergoes simulated movingbed chromatography according to the methods of the present invention inorder to remove host cell protein (“HCP”) and other immaterialcomponents. Preferably affinity chromatography is employed. According toa preferred aspect, a solid phase which comprises Protein A or Protein Gis employed. Any Protein A or Protein G affinity adsorbent may be usedhaving either native or recumbinant Protein A or Protein G with anyconventionally used solid phase backbone (including, e.g., agarose,controlled pure glass, synthetic organic polymer, etc.). According toone aspect the solid phase comprises a recombinant Protein A. Suitablesolid phases include those Prosept rA (Millipore, Bedford, Mass.) andMabSelect (Amersham Biosciences, Uppsala, Sweden). Other solid phasesknown to those of skill in the art for separating an immunoreactivecompound, such as an antibody, from HCP and other immaterial componentsmay be used. In Step E, the product stream is subjected to a low pHincubation step. A holding tank may be conveniently used for this step.Incubation at low pH according to this step is believed to inactivateviruses which may be present in the solution. Step F comprises cationexchange chromatography, preferably with a cation exchange solid phaseusing a bind and elute mode. Suitable cation exchange solid phasesinclude strong cation exchange adsorbents including SP Sepharose XLresins using composite materials such as Hyper D (Biosepra), andtentacle resins (Merck or Tosohaas), and weak cation exchange adsorbentsusing carboxymethyl (CM) ligands. The SMB methods of the presentinvention may be used in this separation step (as well as Step D). StepF is believed to remove leached Protein A (where Step D uses affinitychromatography with a solid phase comprising Protein A) and also toremove viruses if present and other contaminants. Step G comprises anionexchange chromatography, preferably using a strong anion exchange solidphase in a flow through mode. Suitable solid phases include FractogelTMAC. Step G is believed to remove residual contaminants (including DNA)and viruses if present due to adsorption to the anion exchanger. Step Hcomprises a nanofiltration step. Suitable methods include dead endfiltration. Step H is believed to remove viruses which may be present bysize exclusion. Step I comprises an ultrafiltration or diafiltrationstep. Step I is used to adjust concentration of antibody in solution toan appropriate concentration for storage and, if indicated, effectbuffer exchange to an appropriate buffer for storage.

[0051] An alternate process for large scale antibody production isdepicted in FIG. 2. Steps in the process include: (a) Fed batchfermentation in CHO cell culture: yielding 15.000 liter of broth,containing 0.5-2 g/l of antibody; (b) centrifugation for biomassremoval; (c) Protein A chromatography (main objective of this step is toremove the (majority of) host cell proteins (HCP)); (d) cation exchangechromatography (the objective of this step is to remove any Protein Aligand that has leaked from the affinity gel, since both the Protein Aligand and the antibody bind to the gel and fractionation occurs duringselective elution); and (e) anion exchange chromatography (mainobjective is to remove (adsorb) DNA, the MAb is not adsorbed since it isfairly basic in nature).

[0052] 2. Description of SMB Systems

[0053] The present invention relates to methods of using a SMB system aspart of a process for the isolation and purification of immunoreactivecompounds, such as antibodies or antibody fragments produced by cellculture.

[0054] In one aspect of the present invention, a SMB system may be usedto separate an immunoreactive compound from host cell protein and otherimmaterial components employing Protein A affinity resins. In order toaccommodate all steps required for reliable immunoreactive compoundisolation in a Protein A affinity process, an experimental set-up whichcomprises zones for associating immunoreactive compound with solidphase, removing unassociated components, disassociation/elution ofimmunoreactive compound from solid phase andregeneration/re-equilibration of the solid phase are used.

[0055] According to this aspect of the present invention, theimmunoreactive compound is associated with the Protein A module atneutral pH, unassociated components are removed in one or two washsteps, the immunoreactive compound is disassociated/eluted at low pH,and the module is regenerated and re-equilibrated by one, two or threeconsecutive steps. All these steps are continuously performed by a SMBsystem which comprises at least one module which moves through a ringwhich has a plurality of zones.

[0056] According to one aspect of the present invention, the ringconsists of a plurality of zones, suitably from four to eight zones,depending on the number of wash and regeneration/equilibration stepsused. According to the methods of the present invention, a continuousflow of purified immunoreactive compound is obtained, with the amount ofsolid phase and buffer used being substantially reduced in comparisonwith a batch mode. One aspect of the present invention is directed tousing a multi-zone SMB system as described herein for antibodypurification with protein A affinity adsorbents.

[0057] Protein A affinity SMB chromatography may be a powerful way toreduce the substantial cost involved in Protein A affinitychromatography. A reduction in module (or column) volume immediatelytranslates into reduced cost of goods, additionally, smaller modules areeasier to pack and usually cause fewer problems during operation. Afully automated continuous system requires much fewer operatormanipulations, thus reducing the risk for failure.

[0058] According to an alternate aspect of the present invention the SMBsystems described herein can also be applied to the a cation exchangechromatography step in an antibody purification process, which usuallyoccurs after Protein A affinity chromatography step. The economicbenefits of using a SMB system for this step are similar to the ProteinA affinity chromatography step.

[0059] A. General Four (4) Zone SMB Systems

[0060] (i) FIG. 3

[0061]FIG. 3 depicts a four zone SMB system of the present invention.

[0062] According to one embodiment, the feed is applied to two (2)columns in parallel, and makes a second pass though two (2) parallelcolumns. Mass transfer is therefore spread over four columns. A carouselor other similar apparatus may be used to move the columns relative tostationary valves and inlet and outlet streams. The columns moving tothe left from the adsorption zone enter the adsorption wash (or firstwash) zone. The buffer applied here washes unbound material and removesendotoxins. The effluent of the adsorption wash (or first wash) zone isfed back into the adsorption zone to minimize product loss. Theadsorption wash (or first wash) zone consists of two (2) columns inseries thus reducing the amount of buffer required by about 40%. Theproduct is eluted in an elution zone consisting of two (2) columns inseries, again taking advantage of the resulting countercurrent effect.The elution zone is followed by an elution wash zone. The buffer appliedin that zone may be a diluted equilibration buffer, its buffer capacitybeing much lower than the elution zone's buffer capacity. The elutionwash buffer is recycled into the elution zone saving on water cost.

[0063]FIG. 4 depicts a process flow diagram of an alternate embodimentof the SMB system of FIG. 3, a four zone SMB system for the separationof antibody. According to this embodiment, the columns in the adsorptionzone (positions 7 and 8) are connected in parallel to reduce thepressure drop. The amount of adsorbent (solid phase) in the adsorptionzone is dimensioned such that mass transfer is not limiting. Accordingto one aspect of this embodiment, the columns are arranged in acarousel. The rotation time of the carousel is set to meet the requiredcapacity of the feed flow.

[0064] In the system depicted in FIG. 4, the wash (or first wash) zonethe interstitial liquid entering from the adsorption zone is washed outand fed back into the adsorption zone. Most of the endotoxin and hostcell proteins are removed here and end up in the waste stream outflowfrom the adsorption zone. After the wash (or first wash) zone, antibodyis eluted in the elution zone; the columns at positions 3 and 4 comprisean elution zone. The columns at positions 1 and 2 comprise an elutionwash zone and are used to wash out the interstitial eluent which is fedback into the elution zone thus saving eluent. In order to obtain thedesired pH in the elution zone, the elution buffer applied to theelution zone in this embodiment, however, needs to be concentrated.

[0065] (ii) FIG. 5

[0066]FIG. 5 depicts an alternate embodiment of a four (4) zone SMBsystem. This system differs from the one depicted in FIG. 3.

[0067] According to one embodiment, the adsorption zone has 2 times 3columns in parallel, thus slightly increasing the gel utilization. Theadsorption zone, adsorption wash (or first wash) zone, and elution zoneare basically the same as in the system of FIG. 3; however, there is noelution wash zone. This may lead to a decrease in product recovery as asmall amount of eluent will enter the last column of the adsorptionzone. From the product tank a small flow is pumped into a salt/pHgradient zone. This zone establishes a gradual transition in pH and saltconcentration. Keeping the flow rate below the transport rate ofinterstitial liquid (back into the elution zone) assures that there willbe minimal product loss. Presaturating the columns with eluent andproduct will also improve the effectivity of the elution zone.

[0068] An alternate embodiment of the SMB system of FIG. 5 is depictedin FIG. 6. The principle is similar to the systems of FIGS. 3 and 4described above, with the omission of the elution wash zone. However,one additional zone is included in this embodiment, the entrainmentrejection zone. This allows for a higher effluent concentration. Inaddition, this zone will generate a pH profile in the system (in columnpositions 3 and 4). This might give a relatively smooth transition overthe iso-electric point of the protein, thereby minimizing product decay.

[0069] The individual columns used for embodiment depicted in FIG. 6 maybe much smaller than those used in the system of FIG. 3 or FIG. 4. Thesystem volume and gel utilization are also significantly higher. Mainreason for this is that the fraction of the gel volume which is activein the adsorption zone is higher than in the system of

[0070]FIG. 3 or FIG. 4 (6 out of 12 columns instead of 2 out of 4). Theabsolute amount of gel in the adsorption zone is more or less the sameas in the system of FIG. 3 or FIG. 4.

[0071] B. General Eight (8) Zone SMB System

[0072]FIG. 7 depicts an eight (8) zone SMB system of the presentinvention. According to one embodiment, all process steps occur in twopasses. The adsorption and elution zone have two times two parallelcolumns to decrease the linear flow rate. All other zones have 2 columnsin series to take advantage of the countercurrent effect.

[0073] FIGS. 8 to 10 depict alternate embodiments of eight (8) zone SMBsystems of the present invention.

[0074] Example 1 describes a process run using the embodiment of FIG. 8.

[0075] Example 2 describes a process run using the embodiment of FIG. 9.

[0076]FIG. 10 depicts a diagram of an alternate embodiment of an eight(8) zone SMB system. In this embodiment, regeneration of the adsorbenthas been incorporated. The system is sanitized only once per batch run,i.e. once per 15,000 liters. The adsorption zone contains four columns,which are connected as two times two parallel columns. In the first washzone the interstitial fluid is fed back into the adsorption zone. In thefirst wash zone, three columns are connected in series to maximize thebenefits of countercurrent contact. The first wash buffer is introducedin upflow in order to minimize the contact between high saltconcentrations and concentrated MAb solutions. After a column leaves theadsorption zone, the top of the bed contains more concentrated feedsolution than the bottom. In the embodiment of FIG. 10, water savingsare gained from the dilution of first wash zone buffer with the effluentor buffer used for the second wash zone. The second wash zone has twocolumns and is designed to establish a low salt concentration atposition 12 just before it moves to position 11. From the product tank avery low flow of product stream is pumped into column 11 to presaturatethat column before it moves into the elution zone. The only function ofposition 11 is therefore product concentration, the common name for thisprocess step is entrainment rejection. In the elution zone, elutionbuffer is introduced in upflow in two times two columns in parallel. Theelution buffer is diluted with the elution wash buffer used to wash outthe eluent from positions 5 and 6 in the elution wash zone. As aconsequence, the elution buffer applied to the elution zone should beconcentrated in order to compensate for the dilution effect. Columns 1to 4 are used for equilibration and regeneration and comprises anequilibration zone and a regeneration zone. If the regeneration requiresa certain concentration of sodium hydroxide, the regeneration bufferapplied to the system should have a higher concentration to accommodatefor the dilution. This system depicted in FIG. 10 is especially suitedto a carousel type SMB system, which has the flexibility of usingdifferent flow directions in one system.

[0077] To assist in understanding, the present invention will now befurther illustrated by the following examples. These Examples as theyrelate to the present invention should not, of course, be construed asspecifically limiting the invention and such variations of theinvention, now known or later developed, which would be within thepurview of one skilled in the art are considered to fall within thescope of the invention as described herein and hereinafter claimed.

EXAMPLES Example 1 Purification of a Monoclonal Antibody Using an EightZone Simulated Moving Bed System

[0078] The simulated moving bed, SMB, technology was applied to theProtein A initial purification step of a monoclonal antibody. The systemconsisted of twenty columns arranged in a circle attached to a rotatingcarousel. Eight independent zones were established for the SMBcontinuous system.

[0079] A. Description of Equipment

[0080] The central valve, which consisted of three sections (top middleand bottom), delineated the eight processing zones. The top part of thevalve remained stationary during the process and the inter-connectionsof the outlet ports defined the processing zones. The middle section ofthe valve was a Teflon ring that allowed the bottom section to turnfreely. The bottom section of the valve was connected to the top andbottom of each column.

[0081] Each processing zone required a specific buffer and flow rate.Eight HPLC pumps were employed to deliver to the inlet sections of thetop part of the valve a constant fluid flow.

[0082] The rotation of the columns was maintained by a controller thatrotated the carousel at a specific interval. After a specific dwelltime, the carousel rotated one spot to the next position, creating thecounter current flow that reduces buffer consumption and increases resincapacity.

[0083] B. Description of Zones

[0084] In this example twenty columns were attached to the centralvalve. (See FIG. 8) At a snapshot in time, the columns labeled onethrough twenty corresponded, to the following wash zones:

[0085] (i) Equilibration Zone

[0086] Starting with column one, the Equilibration buffer (10 mM EDTA/50mM Tris (base)/0.5M NaCl, pH 7.5) was applied to the column in upwarddirection. The effluent of column one was directed to the bottom ofcolumn two, the effluent of which was capture in sample vessel forfuture analysis.

[0087] (ii) Regeneration Zone

[0088] Regeneration buffer (4M Urea/50 mM Tris(Base) pH 7.0) was beingapplied to column three in the downward direction. The effluent ofcolumn three was directed to the top of column four, the effluent ofwhich was captured in a sample vessel for future analysis.

[0089] (iii) CIP Zone

[0090] Clean in place, CIP zone buffer (1% Phosphoric acid) was appliedin the downward direction to the top of column five. The effluent ofcolumn five column was directed to the top of column six, the effluentof which was captured in a sample vessel for future analysis.

[0091] (iv) Elution Wash Zone

[0092] Purified water (MilliQ) was applied in the upward direction tocolumn seven, the effluent of which was applied to column eight in theupward direction. The effluent of column eight was monitored by a UVmeter and collected in a sample vessel for future analysis.

[0093] (v) Elution Zone

[0094] Elution zone buffer (100 mM Glycine/200 mM Acetic Acid pH 3.5)was applied in an upward direction to both column nine and column ten ina parallel manner. The effluent of column nine and ten were combinedinto a single stream that was then split to wash column eleven andtwelve in an upward manner. The effluent of column eleven and columntwelve were combined to form the product stream, which was monitored bya UV meter and collected in a sample vessel for future analysis.

[0095] (vi) Second Wash Zone

[0096] Second wash zone buffer (50 mM Tris(base)/100 mM Glycine pH 7.5)was applied in a downward direction to column thirteen. The effluent ofcolumn thirteen was applied in a downward flow to column fourteen, theeffluent of which was captured in a sample vessel for future analysis.

[0097] (vii) First Wash Zone

[0098] First wash zone buffer (10 mM EDTA/50 mM Tris(base)/0.5M NaCl)was applied in a downward direction to column fifteen. The effluent ofcolumn fifteen was monitored by a UV meter and then applied to columnsixteen in a downward direction. The effluent of column sixteen wascombined with the Feed stream.

[0099] (viii) Adsorption Zone

[0100] The feed stream, which contained a mixture of host cell proteinand monoclonal antibody and other components, was mixed online with theeffluent of column sixteen. The new stream was then split and applied inparallel to both column seventeen and column eighteen in a downwarddirection. The effluent of column seventeen and column eighteen weremixed and then split and applied in parallel to column nineteen andcolumn twenty in a downward direction. The effluent of column nineteenand column twenty were combined and collected in sample vessel forfuture analysis.

[0101] C. Description of Processing

[0102] The Description of Zones by a column number set forth hereinaboverepresents a snapshot in time. After the specified dwell time hadelapsed, the carousel rotated one step in a clockwise direction. As anexample, this means that column one, which previously was exposed toequilibration buffer in a downward direction was now in the positionthat column twenty was previously occupying. Consequently, column twentywas now located where column nineteen use to be, and so on for theentire column set. From a processing stand point this means that theeffluent of column nineteen and column eighteen were combined then splitand applied in the downward direction to column twenty and column one.The column rotation continued until there is no more feed left toprocess.

[0103] Each zone described in the Description of Zones had a specificpurpose in the processing of the feed. Starting from the CIP zone, theregeneration step was implemented as a process-cleaning step. TheRegeneration Zone follows to ensure that any non-specifically boundcompounds were dissociated from the resin. The Equilibration Zone wasimplemented to displace the Regeneration Zone buffer and to create anenvironment in the column that would permit antibody binding during theinitial loading period. In the Adsorption Zone, the feed streamintroduced the desired product to the column. The First Wash Zonedisplaced the loading feed and washed out non-specific proteins andother impurities. The Second Wash Zone was implemented to bring theconductivity to an acceptable base line. The Elution Zone was created todissociate the antibody from the Protein-A ligand and collect theantibody. The Elution Wash Zone was a test section to check whether allthe antibody was effectively washed out during the Elution Zone.

[0104] Since process economics are dependent on the volume of bufferconsumed during processing, experimentation was performed to determinethe minimal amount of buffer needed to perform the above-mentioned taskassigned to each zone. Table I describes the range of column volumestested for each section. TABLE I Stream Column volumes Regenerant 2.9Strip 2.5 Equliibration 1.9-3.0 Feed 16.7-20.4 Wash 1 3.5 Wash 2 1.9-2.5Elution 0.9-3.9 Elution wash 0.95-1.9 

[0105] B. Results

[0106] A summary of the relative amounts of product, Host cell protein(HCP), and Gentamicin found in the process streams is shown in Table IIbelow. TABLE II Host cell Product protein Gentamicin Stream (%) (%) (%)Depleted feed 4.5 78.3 43.6 Wash 1 effluent NA NA NA Wash 2 effluent 0.40.1 0.01 Eluent/Product 83.8 ˜0.1% 0.03 Eluent wash effluent 0.2 ˜0 ˜0Regenerant effluent NA NA NA Strip effluent ˜0 ˜0.1 ˜0 Equliibrationeffluent NA ˜0.1 NA Total 88.9 78.7 43.6

[0107] A protein recovery of 83.8% was achieved using the SMB processdescribed in this Example. The unaccounted amounts of product are likelyto be found in the regenerant fractions. The antibody concentration inproduct pool was ˜8.6 times higher than in the feed stream. The qualityof the product in terms of monomer content and antibody integrity wasinvestigated by SEC and CE-SDS and shown to have values comparable tobatch Protein-A chromatography

[0108] There was a 3.05 log reduction in HCP (>99.9% removal) and a 3.46log reduction of gentamicin (>99.96% removal) in the elution pool. TheHCP concentration was very similar to results from a conventionalprotein-A chromatography scale-up run. The gentamicin levels were fourtimes lower.

Example 2 Purification of a Monoclonal Antibody Using an Alternate EightZone Simulated Moving Bed System

[0109] A Simulated Moving Bed System which was similar to the SMB systemdescribed in Example 1 was used to purify an antibody. This system alsoconsisted of twenty (20) columns arranged in a circle attached to arotating carousel. Eight independent zones were established for the SMBcontinuous system. A diagram of this system is depicted in FIG. 9.

[0110] The configuration of this system differed from that described inFIG. 8 in (a) that all columns operated in downflow and (b) that theelution wash zone used four (4) columns in series and the elution zoneused two (2) columns in series.

Example 3 Demonstration of Reproducibility of Antibody Purification

[0111] The Simulated Moving Bed System described in Example 2 (anddepicted in FIG. 9) was used to examine the reproducibility of thepresent antibody purification process. In a SMB system reproducibilityis required both in between the individual columns (in this case 20columns), and in between SMB cycles (rotations). Reproducibility inbetween columns is shown in FIG. 11 by comparing the UV traces from Wash1. The detector is depicted in FIG. 9 as UV between columns 15 and 16.The signal from this detector is shown for 20 column switches,representing Wash 1 from 20 individual columns in this particularposition for two independent rotations of the SMB cycle. As can be seenfrom FIG. 11, the signals recorded are consistent from all 20 switchesand even overlap for two independent rotations, thus demonstratingcolumn to column reproducibility of the purification.

[0112] The reproducibility between rotations (of the 20 column system)is shown in FIG. 12. FIG. 12 depicts a plot of antibody concentration(IgG titer), IgG flux, and concentration of host cell proteins (HCP isshown for 9 full rotations of the SMB system. IgG flux (mg/ml) wasdepicted by the top plot of diamonds (♦). IgG titer (mg/ml) was depictedby the lower plot of diamonds (♦). HCP (ppm) was depicted by the plot ofsquares (▪).

I claim:
 1. A method of separating an immunoreactive compound from atleast one immaterial component in a fluid mixture using simulated movingbed chromatography which comprises the steps of: (a) providing asimulated moving bed apparatus that comprises a plurality of modules influid conducting communication, said modules comprising at least onesolid phase; (b) continuously introducing the fluid mixture into saidsimulated moving bed apparatus wherein the fluid mixture contacts thesolid phase in a countercurrent mode; (c) effecting separation of theimmunoreactive compound from at least one immaterial component; and (d)collecting the immunoreactive compound to provide a purified compositionthereof.
 2. A method according to claim 1 wherein said immunoreactivecompound associates with the solid phase to a greater or lesser degreethan at least one immaterial component.
 3. A method according to claim 2wherein the immunoreactive compound associates with the solid phase to agreater degree than at least one immaterial component.
 4. A methodaccording to claim 3 which further comprises the step of effecting saidseparation by contacting said solid phase with an eluent that promotesdisassociation of the immunoreactive compound.
 5. A method according toclaim 4 wherein said solid phase comprises a support material associatedwith Protein A or Protein G.
 6. A method according to claim 5 whereinsaid eluent comprises an acidic buffer.
 7. A method according to claim 1wherein separation is effected using adsorption chromatography,partition chromatography, ion exchange chromatography, size exclusionchromatography or affinity chromatography.
 8. A method according toclaim 7 wherein separation is effected using affinity chromatography. 9.An immunoreactive compound prepared according to the method of claim 1.10. A simulated moving bed (“SMB”) system for separating animmunoreactive compound from at least one immaterial component when bothare present in a fluid mixture, wherein the SMB system incorporates aplurality of zones comprising a solid phase which is contactedcountercurrently by said fluid mixture and wherein said zones comprise:an association zone wherein the immunoreactive compound and at least oneimmaterial component differentially associate with the solid phase; afirst wash zone wherein at least one immaterial component ispreferentially disassociated from the solid phase; and an elution zonewherein the immunoreactive compound is preferentially disassociated fromthe solid phase.
 11. A simulated moving bed system according to claim 10wherein said first wash zone is intermediate between said associationzone and said elution zone.
 12. A simulated moving bed system accordingto claim 10 wherein said solid phase preferentially associates with saidimmunoreactive compound.
 13. A simulated moving bed system according toclaim 12 wherein said solid phase comprises a ligand for affinitychromatography.
 14. A simulated moving bed system according to claim 13wherein said solid phase comprises Protein A or Protein G.
 15. Asimulated moving bed system according to claim 12 wherein said solidphase comprises a cation exchange resin.
 16. A simulated moving bedsystem according to claim 10 which further comprises an elution washzone intermediate between said elution zone and said association zone.17. A simulated moving bed system according to claim 16 which furthercomprises a regeneration zone intermediate between said elution washzone and said association zone.
 18. A simulated moving bed systemaccording to claim 17 which further comprises an equilibration zoneintermediate between said regeneration zone and said association zone.19. A method of separating an immunoreactive compound from at least oneimmaterial component which comprises using the simulated moving bedsystem of claim
 10. 20. A purified immunoreactive compound preparedusing the simulated moving bed system of claim
 10. 21. A method oftreating a patient in need of treatment using an immunoreactive compoundof claim
 20. 22. A method of separating an antibody from at least oneimmaterial component in a fluid mixture where both are present usingsimulated moving bed chromatography which comprises the steps of: (a)providing a simulated moving bed apparatus that comprises a plurality ofmodules in fluid conducting communication, said modules comprising atleast one solid phase which comprises an affinity chromatography ligandwhich preferentially associates with the antibody; (b) continuouslyintroducing the fluid mixture into the simulated moving bed apparatuswherein the fluid mixture contacts the solid phase in a countercurrentmode; (c) effecting separation of the antibody from at least oneimmaterial component; and (d) collecting the antibody to provide apurified composition thereof.
 23. A simulated moving bed (“SMB”) systemfor separating an antibody from at least one immaterial component whereboth are present in a fluid mixture wherein the SMB system incorporatesa plurality of zones comprising a solid phase comprising an affinityresin which is contacted countercurrently by the fluid mixture andwherein said zones comprise: an association zone wherein the antibodyand at least one immaterial component differentially associate with thesolid phase; a first wash zone wherein at least one immaterial componentis preferentially dissociated from the solid phase; and an elution zonewherein the antibody is preferentially disassociated from the solidphase.
 24. A method of separating an immunoreactive compound from atleast one immaterial component in a fluid mixture using simulated movingbed chromatography comprising the steps of: (a) providing a simulatedmoving bed apparatus which comprises at least one module in fluidconducting communication with said apparatus, said module comprising atleast one solid phase and wherein said apparatus comprises a pluralityof zones through which the modules pass; (b) continuously introducingthe fluid mixture into the module in an association zone wherein thefluid mixture contacts the solid phase in a countercurrent mode andwherein the immunoreactive compound associates with the solid phase; (c)continuously introducing a wash buffer into the module comprising theassociated immunoreactive compound in a wash zone wherein the washbuffer contacts the solid phase in a countercurrent mode andsubstantially removes at least one immaterial component from saidmodule; and (d) continuously introducing an elution buffer into themodule comprising the associated immunoreactive compound in an elutionzone wherein the elution buffer contacts the solid phase in acountercurrent mode and whereby the immunoreactive compound issubstantially disassociated from the solid phase; and (e) continuouslyremoving a product stream comprising the immunoreactive compound fromthe module.
 25. A method according to claim 24 wherein said simulatedmoving bed apparatus comprises a plurality of modules.
 26. A methodaccording to claim 24 wherein said solid phase comprises an affinityligand.
 27. A method according to claim 26 wherein said affinity ligandcomprises Protein A or Protein G.
 28. A method according to claim 24wherein said immunoreactive compound comprises an antibody.
 29. Apurified antibody prepared substantially by the method of claim
 28. 30.In a method of purifying an immunoreactive compound from at least oneimmaterial component, the improvement which comprises using simulatedmoving bed affinity chromatography with a solid phase comprising ProteinA or Protein G.